Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Effects of Broodstock Age and Sperm Collection Time over a Natural Spawning Period on Sperm Cryopreservation in Farmed Greenlip Abalone,Haliotis laevigata

This study investigated the effects of broodstock age (2‐ and 3‐yr‐old), and sperm collection time at the beginning, middle, and end of a natural spawning period, on the ability of sperm to tolerate cryopreservation in farmed greenlip abalone, Haliotis laevigata. The quality of sperm was assessed by motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential (MMP), and acrosome integrity (AI). The sperm collected at the middle of a natural spawning period had significantly higher quality than those collected at the beginning and the end of the spawning period in terms of plasma membrane integrity, MMP, and AI of fresh and post‐thaw sperm, post‐thaw sperm motility, and fertilization rate. No significant difference was found between 2‐ and 3‐yr‐old animals in most quality parameters evaluated. The results suggested that sperm collected during the middle of the spawning season should be used for cryopreservation. The efficiency of genetic improvement programs can be further enhanced by using sperm collected from 2‐yr‐old abalone which has a similar ability to tolerate cryopreservation as 3‐yr‐old counterparts.     

A Strategy for Sperm Cryopreservation of Atlantic Salmon,Salmo salar,for Remote Commercial‐scale High‐throughput Processing

Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 10⁷, 3 × 10⁸, and 1 × 10⁹ cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.     

Development of a simple method for sperm cryopreservation of Scombridae fishes in outdoor environments

We developed a simple dry shipper method for cryopreserving the sperm of Scombridae fish in outdoor environments. First, we undertook a preliminary study to optimize the sperm cryopreservation conditions using bullet tuna, Auxis rochei (Risso, 1810) sperm. We found that the optimum cryomedium contained 90% foetal bovine serum (FBS) or 300 mM trehalose as an external cryoprotectant and 10% dimethyl sulfoxide (DMSO) as an internal cryoprotectant. Under these optimized conditions, the post‐thaw sperm had a duration of motility of 500 s and a motility rate of >70%. We then performed practical trials of the optimized protocol in various outdoor environments (e.g., fishing boats and ports) using the sperm of five Scombridae species: chub mackerel, Scomber japonicus (Houttuyn, 1782); blue mackerel, S. australasicus (Cuvier, 1832); skipjack tuna, Katsuwonus pelamis (Linnaeus, 1758); longtail tuna Thunnus tonggol, (Bleeker, 1851) and Pacific bluefin tuna, T. orientalis (Temminck & Schlegel, 1844). The post‐thaw sperm of all five of these species had a duration of motility of 650 s and a motility rate of >70%, indicating that this simple method can be used to obtain high‐quality cryopreserved sperm of various Scombridae species in outdoor environments.     

Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus

We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post‐thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre‐cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to ?50°C at 10.4 ± 0.4°C/min, and immediately immersed in liquid nitrogen. Although this method showed the highest post‐thaw motility in all the conditions we examined, its post‐thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post‐thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post‐thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post‐thaw motility and lowering the cryoprotectant concentration is necessary for commercial‐scale artificial fertilization.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.     

Effects of larval cryopreservation on subsequent development of the blue mussels,Mytilus galloprovincialis Lamarck

In this study, the effects of three commonly used chemicals, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) and their combinations with trehalose, were evaluated on the cryopreservation of D‐larvae of the blue mussel Mytilus galloprovincialis. The larvae were harvested 30 h post‐fertilization at 21 °C and cryopreserved using a standard protocol in 5%, 10% or 15% of DSMO, EG and PG either as single chemical solutions or in combination with 0.2 M trehalose. Among these cryoprotectants, 5% DMSO resulted in the highest post‐thaw survival rate of 55.3±7.8%, although it did not significantly differ from those with 10% and 15% EG. The addition of 0.2 M trehalose did not improve the post‐thaw larval survival rates in all the combinations assessed. The cryo‐effects on subsequent development were evaluated using the D‐larvae frozen with 5% DMSO. The results showed that cryopreservation affected both larval survival and growth in this species. The relative daily mortality rate was significantly higher in treated than control groups over the period from 3 h post‐thaw to day 11 post‐fertilization. On day 6 post‐fertilization, the average larval length in the treated group was significantly smaller than that in the control. From day 11 post‐fertilization, and onwards, differences in these two traits were not significant between treated and control groups. On day 21 post‐fertilization, about 80% of the larvae in both treated and control groups developed eyes and the normalized survival rate in the treated group was 12.5%.     

High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.     

Effect of methanol concentration and thaw rate on the viability and fertility of cryopreserved Arctic char,Salvelinus alpinus (L.), spermatozoa

In two trials, Arctic char (Salvelinus alpinus) semen was frozen in 0.5 mL straws using extenders consisting of 0.3 M glucose and 10%, 12.5% or 15% methanol. Cryopreserved semen was thawed by immersing straws in 25 °C water for 17 s (11.6 °C s^?1) or in 5 °C water for 60 s (3.3 °C s^?1). The viability of the frozen–thawed semen was measured by determining post‐thaw motility and sperm membrane integrity. Two fertility trials were also conducted. There was no effect of trial or thaw rate on post‐thaw sperm viability or fertility. Use of 15% methanol in the extender resulted in the highest overall percentage of sperm motility and fertility. Use of 12.5% methanol as a cryoprotectant resulted in a higher per cent post‐thaw motility and a lower percentage of dead cells than did 10% methanol. Thus, levels of methanol higher than the commonly used 10% are beneficial for cryopreserving Arctic char sperm.     

Dry shipper cryopreservation of seven‐band grouper (Epinephelus septemfasciatus Thunberg) spermatozoa

This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer‐assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post‐thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.     

Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity,cooling rate and thawing temperature

Aquaculture of hard clams Mercenaria mercenaria is a $65 million industry along the east coast and Gulf of Mexico coast in the United States. The goal of this study was to develop a preliminary protocol to cryopreserve trochophore larvae of hard clams. The objectives were to evaluate the: 1) toxicity of cryoprotectants, including dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol and glycerol, at 5, 10, 15 and 20% for exposure time of 1, 15, 30, 45, 60 and 75 min; 2) effects of cooling rates (5, 10, 20 and 30°C/min for the first trial; and 1, 3 and 5°C/min for second trial from 4 to ?80°C), thawing temperature (30, 40 and 50°C) and their interactions on post‐thaw viability. A basic protocol was concluded as: 15‐hr trochophore larvae mixed with DMSO or propylene glycol (5, 10%), equilibrated for 15 min, cooled in a programmable freezer from 4 to ?80°C at a cooling rate of 5°C/min and thawed at 50°C for 6 s. With this protocol, the immediate post‐thaw trochophore survival was 23 ± 14%, and survival to D‐stage was 27 ± 14%. This is the first report on larval cryopreservation in the hard clam and would have application for genetic breeding and seed production.     

Vitrification of sperm from marine fish: effect on motility and membrane integrity

Our goal was to develop a standardized approach for sperm vitrification of marine fish that can be applied generally in aquatic species. The objectives were to: (i) estimate acute toxicity of cryoprotectants over a range of concentrations; (ii) evaluate the properties of vitrification solutions (VS); (iii) evaluate different thawing solutions and (iv) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10‐μL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post‐thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X‐1000^? + 1% Z‐1000^? had an average post‐thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to higher osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.